Studies on the C-terminal tail of Vasopressin 1a receptor

Azam, Maria Tahir (2017). Studies on the C-terminal tail of Vasopressin 1a receptor. University of Birmingham. Ph.D.

Full text not available from this repository.

Abstract

Site-directed mutagenesis and fluorescent-protein based techniques were used to evaluate the role of the C-terminal tail of human V\(_1\)\(_a\) vasopressin receptor (V\(_1\)\(_a\)R). Mutants engineered with the C-tail truncations were characterised with respect to cell-surface expression by enzyme linked immunosorbent assay (ELISA), agonist binding by competition radioligand binding assay and signalling capability by inositol phosphates (InsP-InsP\(_3\)) accumulation assay. A series of Ser/Thr mutations disrupted phosphorylation sites and identified a putative G-protein-coupled receptor kinase 2 (GRK2) consensus site contributing to V\(_1\)\(_a\)R internalisation rate and desensitisation in response to vasopressin. GRK2 and GRK2 constructs mutated at functional domains were used to identify the role of GRK2 in V\(_1\)\(_a\)R internalisation. A V\(_1\)\(_a\)R-mCherry fusion, V\(_1\)\(_a\)R-vYC and A2AR-vYC were generated and likewise functionally characterised. Addition of mCherry or vYC at the C-terminus of V\(_1\)\(_a\)R did not affect the binding affinity of V\(_1\)\(_a\)R, cell-surface expression and ability to signal via Gq/11 coupling. Similarly, A\(_2\)\(_A\)R-vYC displayed WT like binding profile and receptor internalisation. Total internal reflection fluorescence microscopy (TIRF-M) and confocal imaging were performed to monitor V\(_1\)\(_a\)R-mCherry internalisation upon agonist stimulation. Overall, results presented in this thesis provide insight into the role of the C-terminal tail domain in V\(_1\)\(_a\)R internalisation and desensitisation and identify functionally important residues including a putative GRK2 regulatory site.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):